Defibrotide (DF ) is a polydeoxyribonucleotide obtained from controlled depolymerization of DNA from mammalian lungs (Patents). It has been shown to have a profibrinolytic activity (Pescador et al., 1983) and an antithrombotic activity (Niada et al., I98 1 ) and was found to stimulate prostacyclin (PGI?) release (Niada et a[., 1982). Furthermore, DF was demonstrated to be cardioprotective in acute (Niada et al., 1985) and in lethal myocardial ischaemia in the cat (Niada et al., 1986) and in early reperfusion damage (Thiemermann et al., 1985). Moreover, the drug prevented myocardial contracture in ischaemic rabbit heart (Berti et al., 1987). Although D F activity is related to vascular PGI, formation (Lobel & Schror. 1985), the mechanisms of action of the drug are still unknown. Since PGI, synthesis seems to be localized in endothelial cells (MacIntyre et al., 1978), we hypothesized that the increase in PGI, production was due to a beneficial effect of D F on endothelial cell function. An enhancement of endothelial cell metabolism could be related to ADP availability, but also to oxygen delivery. Indeed, we supposed an increase in oxygen release from haemoglobin induced by drug treatment. To verify this hypothesis, we decided to measure the modifications induced by DF on the 2,3-diphosphoglycerate (2,3-DPG) content of erythrocytes. In fact, an increase in 2,3-DPG levels may be an indicator of a reduced affinity of haemoglobin for oxygen. We previously performed some experiments in vitro using a U.V. test (Boehringer, Mannheim, Germany) for the determination of 2,3-DPG in blood. We used peripheral heparinized blood obtained from Wistar (RTIY) anaesthetized rats by puncture at the aorta bifurcation level. The blood was incubated for different times (0, 2.5, 5, 10 and 15 min) at 37°C in the presence of 0.9% (w/v) saline, vehicle of the drug [a placebo solution of citric acid trisodium salt dihydrate at 1% (w/v) in distilled water], or the drug at the concentration of 1.4 mg/ml of blood. As shown in Fig. 1, intra-erythrocyte 2,3-DPG content always significantly ( P < 0.01 by Student's paired t-test) increased in drug-treated samples, with maximal values at 5 and 10 min from incubation. The effect of DF was dose independent, since the increase in drug concentration to 6.4 mg/ml or more did not significantly increase the 2,3-DPG values in the erythrocytes. Our results in vitro seemed to reveal a rapid dissociation of haemoglobin from oxygen due to DF treatment, thus favouring oxygen availability. Treatment with saline or vehicle failed to increase the levels of 2,3-DPG obtained with fresh non-incubated blood. Consequently, we decided to study the modifications induced by DF administration in vivo on 2,3-DPG intraerythrocyte content. We treated Wistar rats for 30 min with the drug i.p. at the dose of 30 mg/kg (which previously was shown able to induce profibrinolytic and antithrombotic activities) or for 1 h orally at the dose of 50 mg/kg, since an oral activity of the drug has also been shown (Niada et a[., 1982). Our results in vivo (Table 1) indicate that the drug, administered i.p. or orally, induces a significant ( P < 0.01) increase in 2,3-DPG intra-erythrocyte levels. The higher availability of oxygen induced by DF could explain the protective effect and the efficacy of the drug during ischaemia, when the presence of oxygen could represent a limiting factor for metabolic cell activity, particularly for endothelial cells. The increase in oxygen consumption by these cells could also favour their use of ADP, which is an inducer of platelet aggregation, thus possibly reducing the aggregation response. The enhancement of metabolic conditions in endothelial cells could promote a stimulated production of PGI, by the same cells. Platelet aggregation is further inhibited by the increase in intracellular cyclic AMP induced by PGI, production. On the basis of these considerations, DF, through its ability to enhance endothelial cell metabolism, could be considered to exert a cytoprotective effect on these cells. In this view, use of the drug during ischaemia may better express its properties. In addition, we have to consider the possibility that DF induces an increased production in vivo of a platelet factor or a plasma factor responsible for stimulating PGI, generation from the cell wall. On the other hand, since it was previously demonstrated (Lobel & Schror, 1985) that PGI, stimulation by DF does not appear to be associated with its fibrinolytic action, we can suppose that the drug acts through different mechanisms in relation to the presence of different drug components each responsible for various activities.
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May 1, 1992
A Wilde + 4
Open Access